Antibodies that bind to α2-antiplasmin crosslinked to fibrin which do not inhibit plasma α2-antiplasmin

ABSTRACT

The present invention relates to a treatment for myocardial infarction and blood clots within a patient, and more specifically to a therapy which enhances clot lysis comprising administering to a patient an antibody directed to α2-antiplasmin crosslinked to fibrin (α2AP-FX) which does not inhibit plasma α2-antiplasmin (α2AP). The invention also relates to a treatment for enhancing clot lysis comprising administering an antibody directed toward α2-antiplasmin crosslinked to fibrin which does not inhibit plasma α2AP together with a thrombolytic agent.

This application is a continuation of application Ser. No. 08/680,634,filed Jul. 16, 1996, now U.S. Pat. No. 5,831,031 which is a divisionalof application Ser. No. 07/980,520, filed Dec. 1, 1992 (U.S. Pat. No.5,582,862, issued Dec. 10, 1996), which is a continuation-in-part ofapplication Ser. No. 07/943,372, filed Sep. 10, 1992 (U.S. Pat. No.5,372,812, issued Dec. 13, 1994), which is a continuation of applicationSer. No. 07/589,003, filed Sep. 27, 1990 (abandoned), which is acontinuation-in-part of application Ser. No. 07/177,222, filed Apr. 4,1988 (abandoned), the contents of which are incorporated herein byreference.

This invention was made with Government support under Contract #HL-02348awarded by the National Institutes of Health. The Government has rightsin this invention.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to a treatment for myocardial infarctionand blood clots within a patient, and more specifically to a therapywhich enhances clot lysis comprising administering to a patient anantibody directed to α2-antiplasmin crosslinked to fibrin (α2AP-Fx)which does not inhibit plasma α2-antiplasmin (α2AP). The invention alsorelates to a treatment for enhancing clot lysis comprising administeringan antibody directed toward α2-antiplasmin crosslinked to fibrin whichdoes not inhibit plasma α2AP together with a thrombolytic agent.

BACKGROUND INFORMATION

The initiating event of many myocardial infarctions (heart attacks) isthe hemorrhage into atherosclerotic plaque. Such hemorrhage oftenresults in the formation of a thrombus (or blood clot) in the coronaryartery which supplies the infarct zone (i.e., an area of coagulationnecrosis which results from an obstruction of blood circulation). Thisthrombus is composed of a combination of fibrin and blood platelets. Theformation of a fibrin-platelet clot has serious clinical ramifications.The degree and duration of the occlusion caused by the fibrin-plateletclot determines the mass of the infarct zone and the extent of damage.

A. Treatment for Myocardial Infarction

The primary goal of current treatment for myocardial infarction involvesthe rapid dissolution of the occluding thrombus and the restoration ofblood flow (“reperfusion”). An agent which is capable of selectivelybinding to and affecting may enhance thrombolysis and may decrease therisk of general hemorrhage to the patient. A successful therapy must becapable of sustained effect so that reformation of the clot does notoccur after the cessation of therapy. If the fibrin-platelet clot isable to reform, then the affected artery may become reoccluded.

The formation of fibrin-platelet clots in other parts of the circulatorysystem may be partially prevented through the use of anti-coagulants(such as heparin). Unfortunately, heparin has not been found to beuniversally effective in preventing reocclusion in myocardial infarctionvictims in which the degree of blood vessel occlusion (the degree of“stenosis”) is greater than or equal to 70%, particularly in thosepatients with severe residual coronary stenosis.

If an individual has formed a fibrin-platelet clot prior to theavailability of medical assistance, the clot may be dissolved throughthe use of thrombolytic agents. A thrombolytic agent is a medicamentcapable of lysing the fibrin-platelet thrombus, and thereby permittingblood to again flow through the affected blood vessel. Such agentsinclude, but are not limited to, streptolinase, prourokinase, urokinase,staphylokinase and tissue-type plasminogen activator (Ganz, W. et al.,J. Amer. Coll. Cardiol. 1:1247-1253 (1983); Rentrop, K. P. et al., Amer.J. Cardiol. 54:29E-31E (1984); Gold, H. K. et al., Amer. J. Cardiol.53:122C-125C (1984)).

B. Mechanism of Fibrin Clot Formation

Clots are composed of both fibrin and blood platelets in various ratios.The fundamental reaction in blood clotting involves the conversion of asoluble plasma protein (fibrinogen) into insoluble fibrin. Theconversion of fibrinogen into fibrin is catalyzed by the enzymethrombin, which is a serine protease. Fibrin chains are crosslinked toeach other by activated Factor XIII. Similarly activated Factor XIIIcrosslinks α2AP to fibrin, concentrating the inhibitor on the clotsurface. These two crosslinking events render the fibrin clot highlyresistant to lysis. The general mechanism of blood clot formation isreviewed by Ganong, W. F. (In: Review of Medical Physiology, 9th ed.,Lange, Los Altos, Calif., pp. 411-414 (1979)). Platelets are disk-shapedstructures present in blood. They contribute to clot formation by boththeir incorporation with fibrin into an insoluble mass and by theirenhancement of the rate of fibrinogen to fibrin conversion and byproviding Factor XIII to enhance fibrin/α2AP crosslinking. Plateletscontribute to clot formation in myocardial infarction and are a majorcomponent of clots that reocclude coronary arteries that have beenreperfused by treatment with a thrombolytic agent.

C. Mechanism of Clot Lysis and Natural Inhibition Thereof

Clot lysis is mediated by plasmin in vivo. Under natural conditions,plasminogen is converted to plasmin by tissue plasminogen activator(t-PA). Activation occurs on the fibrin surface, thus confiningproteolytic activity to the appropriate site. After plasmin is set freeinto the circulation, it is rapidly combined with natural inhibitors.Inactivation of plasmin is the final and necessary step in the processof protecting against undesirable proteolysis. Such plasmin inhibitorsinclude α2-antiplasmin, α2-macroglobulin and α1-antitrypsin, allglycoproteins. α2-antiplasmin has a much higher affinity for plasminthan α2-macroglobulin and binds specifically to plasmin in a 1:1 ratio.The larger pool of α-macroglobulin acts as a reservoir inhibitor. Kane,K. K., Ann. Clin. Lab. Sci. 14:443-449 (1984). Thus, clot lysis by theadministration of plasminogen activators is limited by the rapid andirreversible inactivation of plasmin by plasmin inhibitors.

α2-antiplasmin has three functional domains: the reactive site forplasmin, the plasmin(ogen) or LBS-binding site [complementary to the LBS(lysine-binding site) of plasmin(ogen)], and the cross-linking site forfibrin. Mimuro, J. et al., Blood 69:446-453 (1987). Mimuro et al.disclose antibodies to α2-antiplasmin, one of which (JPTI-1) wasspecific to the reactive site of α2-antiplasmin and prevented formationof α2-antiplasmin-plasmin complexes, thereby inhibiting antiplasminactivity. However, Mimuro et al. do not teach administration of theJPTI-1 antibody to enhance clot lysis. Other antibodies specific forα2-antiplasmin are taught by Plow, E. F. et al., J. Biol. Chem.255:2902-2906 (1980); Wimen, B. et al., Scan. J. Clin. Lab. Invest.43:27-33 (1983); Hattey, E. et al., Thromb. Res. 45:485-495 (1987);Collen, U.S. Pat. No. 4,346,029 (1980); and Collen, U.S. Pat. No.4,198,335 (1980).

D. α2-Antiplasmin Crosslinked to Fibrin

During clotting, α2AP is crosslinked to fibrin by Factor XIIIa. Aoki andcolleagues have demonstrated that this crosslinking of α2AP to fibrin isimportant in preventing the “endogenous fibrinolysis” that occurs whenfibrin-bound plasminogen is activated by fibrin-bound, endogenous,plasminogen activator (Aoki, N. et al., Blood 62:1118-1122 (1983)). Thiscrosslinking may serve as a means of concentrating α2AP at the alphachain site where fibrin appears particularly vulnerable to attack byplasmin (Pizzo, S. V. et al., J. Biol. Chem. 248:4574-4583 (1973)).Plasma clots deficient in crosslinked α2AP undergo a spontaneous lysiswhen suspended in buffer or plasma containing normal amounts of α2AP;the rate of lysis is proportional to the amount of α2AP incorporatedinto the clot (Sakata, Y. and Aoki, N., J. Clin. Invest. 69:536-542(1982)). In a similar fashion, the crosslinking of α2AP to fibrin may beinhibited by a 0.13 mM concentration of a peptide that represents the 12amino-terminal residues of α2AP (Kimura, S. et al., Blood 66:157-160(1985)). This inhibition of crosslinking results in clots that lyse morereadily upon exposure to fibrinolytic agents. In thrombolyticsituations, fibrin-bound α2AP may be the most important inhibitor ofclot lysis; whereas the chief role of soluble α2AP may be to preventcirculating plasmin from degrading other clotting factors.

Work with antibody RWR, has confirmed the importance of α2APcrosslinking in stabilizing the clot against lysis (Reed et al., Proc.Natl. Acad. Sci. USA 87:1114-1118 (1990), Reed et al., Circulation82:164-168 (1990)). For example, in experiments using compressed andwashed plasma clots (to clear away unbound α2AP), RWR alone causes theclots to undergo spontaneous lysis (Reed et al., Proc. Natl. Acad. Sci.USA 87:1114-1118 (1990). This spontaneous lysis is probably due touninhibited plasmin generated by fibrin-associated t-PA's action onfibrin bound plasminogen as has been suggested for α2AP deficiency(Aoki, N. et al., Blood 62:1118-1122 (1983)). In crosslinking to fibrin,α2AP forms a new epitope that is unique to clots.

An antibody that exclusively inhibits α2AP crosslinked to fibrin can beused to target plasminogen activators to a clot as well as to amplifytheir thrombolytic effects. Since antiplasmin crosslinked to fibrin ispresent in small quantities and only at the clot surface, such anantibody is the ideal inhibitor: it prolongs the half-like of plasminproximity to the clot while not interfering with the inactivation ofcirculating plasmin by soluble α2AP. As such it could not cause systemicthrombolysis. Such an agent can induce an ultra-specific α2AP deficiencyat the site of the clot. In doing so, it could augment the normal clotlysis initiated by endogenous t-PA, and reproduce the spontaneousthrombolysis noted in studies of α2AP deficiency. If the antibody weregiven with a clot-specific agent such as t-PA, even more specific lysiswould be obtained than has previously been seen. In addition, because ofits combined specificity for the clot, and its capacity tosimultaneously inhibit fibrin-crosslinked α2AP, this antibody would beextremely useful as a means of targeting plasminogen activators to theclot. Accordingly, the present invention provides antibodies which bindto and inhibit α2AP crosslinked to fibrin, but do not inhibit solubleα2AP.

E. Summnary

In summary, a substantial goal of therapies aimed at treating myocardialinfarction involves limiting necrosis by permitting early reperfusionand by preventing reocclusion. At present, this goal is partiallyachieved through the administration of thrombolytic agents capable ofdissolving the potentially life-threatening fibrin-platelet clots. Thepotential benefit of employing such agents is, however, significantlyoffset by their lack of fibrin specificity (as in the case ofstreptokinase and urokinase), or by their relatively short biologicalhalf-life caused by plasmin inhibitors (which may result in reformationof the fibrin clot, and the accompanying reocclusion of the affectedblood vessels). Hence, a need exists for an improvement in thrombolytictherapy which specifically enhances clot lysis, while minimizingfibrinogen breakdown and preventing reocclusion of the affected coronaryartery.

SUMMARY OF THE INVENTION

It is a general object of the invention to provide a method for treatingmyocardial infarction or a blood clot within a patient.

It is a specific object of the invention to provide a method fortreating myocardial infarction or a blood clot within a patientcomprising administering to the patient a therapeutically effectiveamount of an antibody or fragment thereof capable of bindingα2-antiplasmin crosslinked to fibrin wherein the antibody does notinhibit plasma α2-antiplasmin.

It is another specific object of the invention to provide a method oftreatment for myocardial infarction or blood clots within a patientwhich comprises co-administering to the patient:

(a) an antibody or fragment thereof capable of binding α2-antiplasmincrosslinked to fibrin in a therapeutically effective amount wherein theantibody does not inhibit plasma α2-antiplasmin; and

(b) a thrombolytic agent in an amount sufficient to either (i) dissolvea fibrin-platelet clot or (ii) inhibit the formation of afibrin-platelet clot.

It is a further specific object of the invention to provide a monoclonalantibody or fragment thereof wherein the antibody or fragment thereof iscapable of binding α2-antiplasmin crosslinked to fibrin and does notinhibit plasma α2-antiplasmin.

It is another specific object of the invention to provide a kit usefulfor carrying out the above-described methods, comprising a carrier meansbeing compartmentalized in close confinement to receive two or morecontainer means therein, which comprises:

(1) a first container means containing a therapeutically effectiveamount of the above-described antibody or fragment thereof; and

(2) a second container containing a therapeutically effective amount ofa thrombolytic agent.

Further objects and advantages of the present invention will be clearfrom the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Binding of monoclonal antibodies to clots in the presence of anexcess of soluble α2-antiplasmin.

FIG. 2. Binding of α2AP-Fx monoclonal antibodies to fibrinogen.

FIG. 3. Clot lysis by α2AP-Fx monoclonal antibodies.

FIG. 4. Inhibition of soluble α2AP by α2AP-Fx monoclonal antibodies.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method for treating myocardialinfarction or a blood clot within a patient comprising administering tothe patient a therapeutically effective amount of an antibody orfragment thereof capable of binding α2-antiplasmin crosslinked to fibrinwherein the antibody does not inhibit plasma α2-antiplasmin.

The present invention also involves a method of treatment for myocardialinfarction or blood clots within a patient which comprisesco-administering to the patient: (a) a therapeutically effective amountof an antibody or fragment thereof capable of binding α2-antiplasmincrosslinked to fibrin wherein the antibody does not inhibit plasmα2-antiplasmin; and (b) a thrombolytic agent in an amount sufficient toeither (i) dissolve a fibrin-platelet clot or (ii) inhibit the formationof a fibrin-platelet clot.

Blood clots which may be treated according to the methods of theinvention include, but are not limited to pulmonary thromboembolism,deep venous thrombosis, cerebral embolism, renal vein and peripheralarterial thrombosis, and the like.

Antibody fragments include F(ab′)₂ or F(ab) molecules, as well as anyfragment capable of binding to α2-antiplasmin crosslinked to fibrinwherein the antibody fragment does not inhibit plasma α2-antiplasmin.Preferably, the antibody or fragment thereof is substantially purifiedand/or isolated.

The antibodies or fragments thereof of the present invention can bemonoclonal antibodies or fragments thereof. It is preferable to employthe F(ab′)₂ fragment of such an antibody for this purpose, in order tominimize any immunological reaction caused by the Fc portion of theimmuno-globulin. Procedures for preparing monoclonal antibodies aredisclosed by Kaprowski, H. et al. (U.S. Pat. No. 4,172,124); and Kohleret al. (Nature 256:495-497 (1975)). More specifically, the preparationof monoclonal antibodies capable of preventing the inhibition of plasminare taught by Mimuro, J. et al., Blood 69:446453 (1987).

Another method for obtaining the monoclonal antibodies of the presentinvention is to use peptide immunogens which mimic the uniquecrosslinked α2AP-Fx epitope. For example, it is possible to synthesize apeptide containing the α2AP-Fx crosslink as shown below:

    Asn-Gln-Glu-Gln-Val-Ser-Pro (α2AP 1-7)          |Thr-Trp-Lys-Pro-Gly-Ser-Ser (a chain 301-3307)

To synthesize this crosslinked sequence, the combined Boc and F-mocstrategy would be used. A peptide that imitates the gamma-gamma chaincrosslink of fibrin has been used successfully as an immunogen togenerate antibodies that specifically recognize the gamma-gammacrosslink site in fibrin (Matsueda, G. R. et al., FASEB J. 2:A1411(1988)). Once synthesized, the α2AP-fibrin crosslink peptide will becoupled to keyhole limpet hemocyanin. It will then be used as animmunogen. In a preferred embodiment, the antibody or fragment thereofis capable of binding a peptide comprising the α2AP-Fx crosslink asshown below:

    Asn-Gln-Glu-Gln-Val-Ser-Pro (α2AP 1-7)          |Thr-Trp-Lys-Pro-Gly-Ser-Ser (a chain 301-3307)

The invention also provides hybridomas which are capable of producingthe above-described antibodies. A hybridoma is an immortalized cell linewhich is capable of secreting a specific monoclonal antibody.

The term “thrombolytic agent” is meant to refer to any agent capable ofeither dissolving a fibrin-platelet clot, or inhibiting the formation ofsuch a clot. Examples of thrombolytic agents include streptokinase,prourokinase, urokinase, and tissue-type plasminogen activator. Use oft-PA for these purposes is especially preferred. Although natural t-PAmay be employed, it is preferable to employ recombinant t-PA. Theinvention may additionally employ hybrids, physiologically activefragments or mutant forms of the above thrombolytic agents. The term“tissue-type plasminogen activator” as used herein is intended toinclude such hybrids, fragments and mutants, as well as both naturallyderived and recombinantly derived tissue-type plasminogen activator.

By the term “co-administered”, it is intended that each of the antibodyor fragment thereof and thrombolytic agent will be administered during atime frame wherein the respective periods of pharmacological activityoverlap. The two agents can be administered simultaneously orsequentially.

As stated, the methods of the invention comprise the administration ofthe antibody or fragment thereof alone or in combination with athrombolytic agent. When administered alone the antibody may enhance invivo thrombolysis by significantly augmenting clot lysis by endogenousplasminogen activators. Alternatively, the antibody or fragment thereofis administered with a thrombolytic agent. In this embodiment, theantibody or fragment thereof and the thrombolytic agent of the presentinvention are intended to be co-administered to the recipient. It ispreferable to provide the antibody or fragment thereof to the patientprior to the administration of the thrombolytic agent. It is mostpreferable to provide the antibody or fragment thereof 45 minutes,preferably 30 minutes, prior to the administration of the thrombolyticagent.

When used alone, an amount of the antibody or fragment thereof capableof enhancing clot lysis when provided to a patient is a “therapeuticallyeffective” amount. In order to enhance clot lysis and prevent clotreformation, it is desirable to provide between 3 to 100 nmole ofantibody or fragment thereof per kilogram of patient weight, and mostpreferably between 3 to 6 nmole of antibody or fragment thereof perkilogram of patient weight. This dosage may be administered, in oneembodiment, over a period of between 60 to 480 minutes, by continualintravenous infusion at a rate of 0.10-1.0 mg/kg min. Alternatively, itis possible to provide the antibody or fragment thereof in anintravenously injectable bolus at a dose of between 3 to 100 nmole/kg,and most preferably between 3 to 6 nmole (of antibody or fragmentthereof) per kilogram of patient weight. If the antibody or fragmentthereof is provided in this manner, a single bolus is sufficient toprevent potential clot reformation. The antibody or fragment thereof ofthe present invention may be dissolved in any physiologically toleratedliquid in order to prepare an injectable bolus. It is preferable toprepare such a bolus by dissolving the antibody or fragment thereof innormal saline.

When the antibody or fragment thereof is co-administered with athrombolytic agent, it is desirable to provide between 3 to 100 nmole ofantibody or fragment thereof per kilogram of patient weight, and mostpreferably between 3 to 6 nmole of antibody or fragment thereof perkilogram of patient weight. This dosage may be administered, in oneembodiment, over a period of 60 to 480 minutes, by continuousintravenous infusion. Alternatively, it is possible to provide theantibody or fragment thereof in an intravenously injectable bolus at adose of between 3 to 100 nmole/kg, more preferably 3 to 6 nmole/kg, andmost preferably between 1 to 3 nmole/kg of patient weight. An amount ofthrombolytic agent capable of causing such lysis is a “therapeuticallyeffective” amount. The thrombolytic agent of the present invention ispreferably provided at a dose of between 0.5 to 1.0 mg per kg of patientweight. In one embodiment, the thrombolytic agent is provided over aprolonged period (i.e., from about 180 to about 1440 minutes). In apreferred embodiment, the thrombolytic agent of the present invention isprovided as an intravenously injected bolus containing between 0.5 to1.0 mg/kg, and most preferably between 0.5 to 0.75 mg/kg. Thethrombolytic agent of the present invention may be dissolved in anyphysiologically tolerated liquid in order to prepare an injectablebolus. It is, however, preferable to prepare such a bolus by dissolvingthe thrombolytic agent in normal saline.

A patient treated according to the preferred embodiment will, therefore,receive an intravenously injected bolus of the antibody or fragmentthereof in combination with an intravenously injected bolus of thethrombolytic agent. This preferred treatment minimizes the amount oft-PA required for thrombolysis, thus reducing the extent of fibrinogenbreakdown and lessening any tendency for general hemorrhage.Importantly, the use of the preferred treatment results in thedissolution of the occluding thrombus at a rate which greatly exceedsthe rate of thrombus dissolution when either the antibody (or fragmentthereof) or the thrombolytic agent is provided by infusion.Additionally, the risk of reocclusion is substantially reduced.

As would be apparent to one of ordinary skill in the art, the requireddosage of the antibody (or fragment thereof) or thrombolytic agent willdepend upon the severity of the condition of the patient, and upon suchcriteria as the patient's height, weight, sex, age, and medical history.

The antibody (or fragment thereof) or thrombolytic agent of the presentinvention can be formulated according to known methods to preparepharmaceutically useful compositions, such as by admixture with apharmaceutically acceptable carrier vehicle. Suitable vehicles and theirformulation are described, for example, in Remtington's PharmaceuticalSciences (16th Ed., Osol, A. (ed.), Mack, Easton Pa. (1980)). In orderto form a pharmaceutically acceptable composition suitable for effectiveadministration, such compositions will contain an effective amount ofthe antibody (or fragment thereof) or thrombolytic agent, either alone,or with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achieved bythe use of polymers to complex or absorb the antibody (or fragmentthereof) or thrombolytic agents of the present invention. The controlleddelivery may be exercised by selecting appropriate macromolecules (forexample, polyesters, polyamino acids, polyvinyl pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine sulfate). The rate of drug release may also be controlled byaltering the concentration of such macromolecules. Another possiblemethod for controlling the duration of action comprises incorporatingthe therapeutic agents into particles of a polymeric substance such aspolyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylenevinylacetate copolymers. Alternatively, it is possible to entrap thetherapeutic agents in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,by the use of hydroxymethylcellulose or gelatin-microcapsules orpoly(methylmethacrylate) microcapsules, respectively, or in a colloiddrug delivery system, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, nanocapsules, or in macroemulsions. Suchteachings are disclosed in Remington's Pharmaceutical Sciences (1980).

The antibody (or fragment thereof) or thrombolytic agent may be providedto a patient by means well known in the art. Such means of introductioninclude oral means, intranasal means, subcutaneous means, intramuscularmeans, intravenous means, intra-arterial means, or parenteral means. Inthe most preferred method of treatment for myocardial infarction, apatient is provided with a bolus (intravenously injected) containingbetween 0.5 to 1.0 mg/kg.

By virtue of their recognition of α2AP crosslinked to fibrin, theantibodies of the present invention will be thrombus-specific, and areuseful for targeting plasminogen activators to the clot; the diagnosticdetection of thrombi; inhibiting the reversible crosslinking of α2AP tofibrin and thus should secondarily increase thrombolysis; and these MAbsshould also not interfere with the inactivation of plasmin by α2AP, andthus markedly amplify the effects of endogenous or administeredplasminogen activators.

The present invention also provides a method of detecting the presenceof a clot (more specifically, a fibrin-platelet clot) in a biologicalsample, comprising: (a) contacting the sample with the above-describedantibody wherein the antibody is specific for an antigenic determinantcharacteristic of the clot, under conditions such that binding of theantibody to the antigenic determinant occurs, and (b) detecting thepresence of the antibody bound to the antigenic determinant, the bindingbeing related to the presence of a clot in the sample.

The present invention further provides a method of diagnosing thepresence of a clot in a patient comprising administering to the patienta conjugate comprising: (a) the above-described antibody or fragmentthereof, linked directly or indirectly to (b) a moiety capable of beingdetected by a source external to the patient, and detecting the presenceof the detectable moiety.

The present invention is described in further detail in the followingnon-limiting examples.

EXAMPLE 1 Preparation of α2AP-Fx Immunogen

Typically 100 to 1000 ml of pooled plasma was clotted with 20 mM CaCl₂and 100 units of thrombin overnight at 37° C. The clot was washed withTBSA containing 2 mM CaCl₂, 100 mM EACA and 1 mM iodoacetamide until theA280 was less than 0.03. Then 1 mg of thermolysin in 10 ml of 0.1 M NaBorate pH 7.2 with 2 mM CaCl₂ was added. The clot was incubated at 37°C. until it was visibly digested. Then it was centrifuged at 4000 rpmfor 15 minutes and the supernatant was filtered through a 5 μM filter.Subsequently, it was incubated with polyclonal rabbit anti-α2AP agarose,or RWR agarose for 2 hours at room temperature. After washing with PBSwith 0.5 M NaCl until the A280 was <0.02, the bound α2AP-Fx fragmentswere eluted with 0.2 M glycine pH 2.8 and neutralized with 1.0 M Tris,pH 8.0.

EXAMPLE 2 Generation of Monoclonal Antibodies

Mice were repeatedly immunized subcutaneously with approximately 50 μgof purified α2AP-Fx antigen. Somatic cell fusion was performed asdescribed (Reed et al. Proc. Natl. Acad. Sci. USA 87:1114-1118 (1990)).Hybridomas showing binding were then rescreened for their ability tobind to α2AP crosslinked to fibrin in washed clots in the presence of a10-fold excess of plasma supernatant containing soluble antiplasmin asan inhibitor. After washing the clots and removing the plasmasupernatant, bound antibody was detected by the addition of ¹²⁵I-goatantimouse antibody. MAbs showing no significant inhibition by plasmawere selected for further study. FIG. 1 demonstrates that these MAbs(1F2, 3G2, 7D3) bound to clots at a level comparable to that of aspecific antifibrin antibody (59D8) and to a much greater extent thandid the antibody to both soluble and fibrin crosslinked α2-antiplasmin(RWR). Under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Micro-organisms for the Purposes of PatentProcedure, a hybridoma cell line producing monoclonal antibody 1F2 wasdeposited with the following International Depository Authority:American Type Culture Collection, now located at 10801 University Blvd.,Manassas, Va. 20110-2209 USA. The deposit was made on Jul. 11, 1996, andgiven the accession number ATCC HB-12149. Under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for the Purposes of Patent Procedure, a hybridoma cellline producing monoclonal antibody 3G2 was also deposited with thefollowing International Depository Authority: American Type CultureCollection, now located at 10801 University Blvd., Manassas, Va.20110-2209 USA. The deposit was made on Jul. 11, 1996, and given theaccession number ATCC HB-12148.

To verify that the MAbs obtained did not bind to fibrinogen solid phasebinding experiments were performed (FIG. 2). Wells of a microtiter platewere coated with purified fibrinogen or a control antigen. The hybridomasupernatant was then incubated in these wells for one hour. Afterwashing the amount of bound antibody was determined by the addition of¹²⁵I-goat antimouse antibody. The specific binding of these antibodiesto fibrinogen was compared to a positive control (antifibrinogen MAb4A5, and antifibrin 59D8) and a negative control MAb (RWR). These MAbsappeared to show little or no significant binding to this fibrinogenpreparation.

Clot lysis assays were performed to determine whether these MAbs couldinhibit α2AP crosslinked to fibrin. Plasma (100 μl) was clotted withtrace amounts of radiolabelled fibrinogen. Then hybridoma supernatants(in triplicate) were added to each clot with 1 unit of t-PA. The clotswere incubated overnight and the amount of lysis was compared to RWR(positive control) and buffer alone (negative control). Compared to thenegative control, hybridomas 1F2, 4H6 and 3G2 accelerated clot lysis atlevels which were comparable to the other MAb RWR (FIG. 3).

Given that the MAbs appeared to enhance clot lysis by inhibitingα2AP-Fx, it was next tested whether they could inhibit soluble α2AP inplasma. A standard α2AP assay was performed essentially as previouslydescribed using the Stachrom kit (Reed et al., Circulation 82:164-168(1990)), i.e., residual α2-antiplasmin activity was measured by achromogenic substrate assay kit from Stachrom (Asnières, France), exceptthat hybridoma culture supernatants were added as inhibitors. FIG. 4shows that RWR significantly inhibited the α2AP in plasma but 3G2 and1F2 had no significant inhibitory effect on soluble α2AP.

All publications mentioned hereinabove are hereby incorporated in theirentirety by reference.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

What is claimed is:
 1. A monoclonal antibody or fragment thereof whereinsaid antibody or fragment thereof is specific to an epitope formed bythe crosslinking of α2-antiplasmin to fibrin and inhibits α2-antiplasmincrosslinked to fibrin so as to increase endogenous fibrinolysis but doesnot interfere with the inactivation of circulating plasmin by solubleα2-antiplasmin.
 2. A kit useful for carrying out a method of treatmentfor myocardial infarction or blood clots within a patient whichcomprises co-administering to said patient: (a) an antibody or fragmentthereof which is specific to an epitope formed by the crosslinking ofα2-antiplasmin to fibrin and inhibits α2-antiplasmin crosslinked tofibrin so as to increase endogenous fibrinolysis but does not interferewith the inactivation of circulating plasmin by soluble α2-antiplasmin,in a therapeutically effective amount; and (b) a thrombolytic agent inan amount sufficient to either (i) dissolve a fibrin or fibrin-plateletclot or (ii) inhibit the formation of a fibrin or fibrin-platelet clot,comprising a carrier means being compartmentalized in close confinementto receive two or more container means therein, which comprises: (1) afirst container means containing a therapeutically effective amount ofsaid antibody or fragment thereof (a); and (2) a second containercontaining a therapeutically effective amount of said thrombolytic agent(b).
 3. The kit according to claim 2, wherein said thrombolytic agent isselected from the group consisting of streptokinase, prourokinase,urokinase, staphylokinase and tissue-type plasminogen activator.
 4. Thekit according to claim 3, wherein said thrombolytic agent is tissue-typeplasminogen activator.
 5. The monoclonal antibody or fragment thereof ofclaim 1, wherein said antibody or fragment thereof is generated byimmunizing an animal with α2-antiplasmin crosslinked to fibrin.
 6. Thekit of claim 2, wherein said antibody or fragment thereof (a) isgenerated by immunizing an animal with α2-antiplasmin crosslinked tofibrin.
 7. A monoclonal antibody or fragment thereof wherein saidantibody or fragment thereof is specific to an epitope formed by thecrosslinking of α2-antiplasmin to fibrin and inhibits α2-antiplasmincrosslinked to fibrin so as to increase endogenous fibrinolysis, andwherein the mean inhibition of soluble or plasma α2-antiplasmin by saidantibody or fragment thereof, as measured using a chromogenic substrateassay, is about equal to or less than that of monoclonal antibody 1F2(ATCC HB-12149).
 8. The monoclonal antibody or fragment thereof of claim7 wherein said antibody or fragment thereof is generated by immunizingan animal with α2-antiplasmin crosslinked to fibrin.
 9. The kit of claim2, wherein the mean inhibition of soluble or plasma α2-antiplasmin bysaid antibody or fragment thereof (a), as measured using a chromogenicsubstrate assay, is about equal to or less than that of monoclonalantibody 1F2 (ATCC HB-12149).
 10. The kit of claim 9, wherein saidantibody or fragment thereof (a) is generated by immunizing an animalwith α2-antiplasmin crosslinked to fibrin.